Significance of vasculopathy in IgA nephropathy patients with regard to Oxford classification and immunostaining findings: a single center experience

Introduction: immunoglobulin A nephropathy is the most common primary glomerulonephritis around the world. It remains poorly understood whether vascular pathology plays an important role in the progression of IgAN. Objectives: Since very few studies regarding vascular disorders in IgAN are published, we aimed to evaluate the role of vascular disease in a group of IgAN patients, especially with regard to Oxford classification, immunostaining findings and various laboratory and clinical data.Patients and Methods: The study was conducted from July 2009 to March 2013. All kidney biopsies were prepared for light and immunofluorescence microscopy. Results: Of the 136 patients, 94 (69%) were male. Of the 136 kidney biopsies, 37 patients had crescents, 2 had lesions of thrombotic microangiopathy (TMA), 10 had fibrinoid necrosis and one patient had morphologic lesions of small vessel vasculitis (with negative ANCA). In this study, there was no significant difference in scores of arteriolosclerosis and intimal fibrosis between males and females. There was a significant correlation between scores of arteriolosclerosis with serum creatinine. This correlation for scores of intimal fibrosis of interlobular artery was also significantly positive. The correlation of arteriolosclerosis with amount of proteinuria was significantly positive too. Conclusion: The association of vasculopathy with serum creatinine and amount of proteinuria further support the role of vasculopathy in aggravation of IgAN. We also had different results with regard to the prevalence of TMA in previous published studies, which needs further investigation in larger series of IgAN patients.


Introduction
Immunoglobulin A nephropathy (IgA) is the most common primary glomerulonephritis around the world (1). Patients with (IgAN) have variable clinical courses and during the past decade much attention has been focussed toward factors having prognostic significance (1,2). Various morphologic lesions have been assessed for predicting progression of IgAN. However, after publication of Oxford classification of IgAN, four morphologic lesions, known as MEST variables, have been selected for the prediction of renal prognosis of IgAN: mesangial hypercellularity (M), endocapillary proliferation (E), segmental sclerosis or adhesion (S), and tubular atrophy/interstitial fibrosis (T) (3,4). Many investigations have focused on risk factors for kidney insufficiency in IgAN. Whether, other renal morphologic variables may have prognostic significance, Journal of Renal Injury Prevention, Volume 2, Number 2, June 2013 is of much interest recently. Presence of extracapillary proliferation (1)(2)(3), location of deposited immunoglobulins (mesangial versus mesangio-capillary), have been suggested as having prognostic significance (1-3). However, it remains poorly understood whether vascular pathology plays an important role in the progression of IgAN. In a study to evaluate the role of vascular disease in the progression of IgAN by morphometric analysis in 71 IgAN patients, Katafuchi et al. observed that hypertension and vessel area were equally important as predictors of glomerular sclerosis and IgAN progression (4). A recent study in Japan, revealed that histological grade involving also vasculopathy at the initial biopsy was found to be a reliable parameter to predict IgAN progression (5,6).

Objectives
Very few studies regarding vascular disorders in IgAN are published. We therefore aimed to assess the role of vascular disease in a group of IgAN patients, especially with regard to Oxford classification, immunostaining findings and various laboratory and clinical data.

Patients and Methods
This study was conducted according to the Oxford classification of IgAN (1,4) from July 2009 to March 2013.

Definition of IgAN
The pathologic diagnosis of IgAN necessitates the observation of IgA-dominant mesangial or mesangialcapillary immune deposits through immunofluorescence (IF) microscopy in the absence of C1q deposits (1,2). The immune deposits were semi-quantified from 0 to 3+ positive bright. The biopsy specimens were sent to a reference laboratory. None of the patients underwent treatment before the biopsy. Those biopsies that contained less than 8 glomeruli were excluded from this study. None of the patients had a history of collagen vascular diseases or liver cirrhosis.

Histologic data
All kidney biopsies were prepared for light and direct IF microscopy. First, we performed an IF review, then we evaluated the glass slides. After an IF diagnosis of IgAN, we reviewed the histopathology glass slides to describe the four morphologic variables of Oxford classification and other morphologic lesions.

Definitions of morphologic variables of IgAN and MEST (Oxford classification)
We estimated the presence of mesangial hypercellularity (M), endocapillary proliferation (E), segmental glomerulosclerosis (S) and the proportion of tubular atrophy and interstitial fibrosis, IF/TA (T), as defined in the Oxford classification (1-3).

Vascular definitions
Intimal thickening was scored by comparing the thickness of the intima to that of the media in the same segment of vessel. Intima was scored variously as normal (score: 0), and thickened to more or less than the thickness of the media (score: 2 or 1, respectively). Arteriolosclerosis (arteriolar hyaline) was noted as the proportion of arterioles affected and was scored according to the following categorization; normal arterioles (score: 0), 1-25% affected arterioles (score: 1), 26-50% affected arterioles (score: 2) and >50% affected arterioles (score: 3) (1-3). The scoring criteria are illustrated in Figure 1.

Clinical and laboratory data
We reviewed the patients' medical records to obtain various demographic, clinical and laboratory data at the time of their biopsies and for follow-up. We gathered the following data at the time of biopsy: race, gender, age, serum creatinine and proteinuria (based on a 24-hour urine collection).
Ethical issues (1) The research followed the tenets of the Declaration of Helsinki; (2) informed consent was obtained; and (3) the research was approved by the institutional review board.

Statistical analysis
We determined the frequency, mean values and standard deviations of the categorical and continuous variables. The statistical significance of the differences between the male and female groups was done using the Mann-Whitney U test. We used the Spearman's correlation coefficient to check the correlations. We used a computer program (SPSS version 16.0, Chicago, IL) for statistical analysis. P<0.05 was considered statistically significant.

Population Characteristics
In our observational study, we enrolled a total of 136 IgAN patients' biopsies.

Prevalence
Of the 136 patients, 94 (69%) were male. The mean age of the patients was 37.6±13 years. The morphologic variables of the Oxford classification are illustrated in Table 1. The mean proteinuria was 1725±1247 mg/day (median=1500 mg/day). The mean number of glomeruli in all renal biopsies was 14.8±7.3. Table 2, shows the frequency and intensity scoring of antibody deposition. In all biopsies, the mean number of totally sclerotic glomeruli number was 2.5±3.2 (median=2). Also, the mean of serum creatinine was 1.6±1.5 mg/dL (median=1.2 mg/dL). Of the 136 kidney biopsies, 37 (27.2%) patients had crescents, 2 (1.5%) patients had lesions of thrombotic microangiopathy (TMA), 10 (7.3%) had fibrinoid necrosis and one (0.7%) patient had morphologic lesions of small vessel vasculitis (with negative ANCA). Tables 3 and 4 show, the frequency distribution of scores of arteriolosclerosis and intimal fibrosis of interlobular arteries.

Gender differences
In this study, there was no significant difference in the scores of arteriolosclerosis and of intimal fibrosis between males and females (p>0.05).

Correlations
There was significant correlation between scores of http://journalrip.com Journal of Renal Injury Prevention, Volume 2, Number 2, June 2013 arteriolosclerosis with serum creatinine (r= 0.291, p= 0.001). This correlation for scores of intimal fibrosis of interlobular artery was also significantly positive (r=0.339, p<0.001). While, there was no significant association between proteinuria and scores of intimal fibrosis of interlobular arteries (P= N.S), the correlation of arteriolosclerosis with amount of proteinuria was significantly positive (r= 0.192, p<0.025). Table 5 shows the association of morphologic variables of Oxford classification with scores of arteriolosclerosis and intimal fibrosis of interlobular arteries. Similarly, Table 6 shows the association of deposited antibodies intensity score with scores of arteriolosclerosis and intimal fibrosis of interlobular arteries. We did not correlate the other vascular lesions, such as TMA and small vessel vasculitis, with various clinical, laboratory and pathological data because the numbers of cases were very small.

Discussion
The mechanism of IgAN progression is still ill-understood (1,7). Predictors for poor outcome comprise both clinical and histological findings. However, these factors may be better indicator of irreversible damage than the activity of the disease. The presence of kidney dysfunction, proteinuria and high blood pressure at the time of renal biopsy have been considered to be associated with the disease progression and correlated with interstitial fibrosis/tubular atrophy too (7)(8)(9). In a study on fifty-one patients with biopsy-proven IgAN, who were followed for 24 months, Brabcova et al. found that higher transforming growth factor-β1 (TGF-β1) and severe chronic vasculopathy were associated with the IgAN progression 24 months after biopsy. They suggested that, besides the known risk factors for chronic renal failure, advanced vasculopathy was associated with the IgAN progression (10). In our study, significant correlation between scores of arteriolosclerosis and intimal fibrosis of interlobular arteries with serum creatinine was observed. Also, the correlation of arteriolosclerosis with level of proteinuria was significantly positive. We also found, the significant positive association of arteriolosclerosis with S variant of Oxford classification and also a significant positive correlation of scores of interlobular arteries with scores of C3 deposition intensity. It is remarkable to notice that chronic vasculopathy, causes renal tissue ischemia and resultant, angiotensin II-dependent TGF-β1 overexpression, which was identified to be a critical component of renal fibrogenesis (10)(11)(12). TGF-β1 is a potent stimulus of the fibrosis leading to interstitial damage which is an important factor in severity of kidney injury and progressive renal failure (10)(11)(12). TMA occurs in IgAN, however, it is uncommon in the setting of this disease (13). The study published by El Karoui et al. in Paris, showed that 53% of

Conclusion
The association of vasculopathy with serum creatinine and level of proteinuria support the role of vasculopathy in aggravation of IgAN. We also found a very low prevalence of TMA as compared with a French study, which needs more studies in larger series of IgAN.
Author's contributions MM and HN wrote the manuscript equally.  Table 2. Frequency of antibody and complement deposition intensity score.